Laboratory Animal Science Vol 48, No 6 Copyright 1998 December 1998 by the American Association for Laboratory Animal Science Mouse Models of Atherosclerosis Jonathan D. Smith In the beginning of this decade the Nobel laureate David A collection of these macrophage foam cells makes up the Baltimore predicted that the 1990s would be the decade of first grossly visible lesion, called the fatty streak. The next the mouse, and the enormous bounty of biological informa- stage of lesion development is called the intermediate fibrous tion gained through the use of genetically modified mice plaque, which involves the proliferation of arterial smooth has turned his prediction into reality. muscle cells forming a cap over the fatty streak. The cells of Coronary heart disease is the most common cause of the foam-cell core often die, leading to the deposition of ex- death in the United States. Most cases are associated with tracellular cholesterol crystals. The advanced complex le- atherosclerosis, often initiated by hypercholesterolemia. sion is one that involves a thrombotic event, often due to the Atherosclerosis is a complex disease with both genetic and rupture of the plaque surface, exposing the highly environmental factors. Family history is a significant risk thromobogenic subendothelial surface, an event that can lead factor for cardiovascular disease, although the specific ge- to acute myocardial infarction. This progression of an ath- netic factors that lead to atherosclerosis susceptibility have erosclerotic lesion is not fast but rather develops over many been difficult to ascertain, as the vast majority of cases are years, and can be initiated during childhood and adolescence. not associated with a monogenic disorder like familial hy- The following is a list of questions that can be used to judge percholesterolemia, which is caused by a mutation in the the usefulness of animal models of atherosclerosis: (1) What low-density lipoprotein (LDL) receptor gene. Most cases of is the nature of the experimental lesions and their similarity atherosclerosis are thought to be associated with polygenic to human lesions; (2) is the plasma lipoprotein profile and factors, with variations in several minor genes coming to- metabolism similar to metabolism in humans; (3) what is the gether and interacting with the environment to create sus- time frame necessary for lesions to form, and how long does it ceptibility to this condition. As Robert Wissler has described take to breed the animals for the studies; (4) what is the cost in the introduction, it is practically impossible to study this of acquiring and maintaining the animals; (5) what is the abil- disease well in humans, as it is difficult to sort out the ge- ity to perform in vivo manipulations and imaging; and (6) netic and environmental components. Long-term dietary what is the ability of the model to take advantage of classical studies are obviously impossible to perform in humans. and molecular genetic approaches? Atherosclerosis develops slowly with age, and is often as- The mouse as a model meets many of these criteria, but certained only after a myocardial infarction, which can first it is important to acknowledge many important dif- mask the assignment of “affected” versus “unaffected” in ferences between mice and humans. The average lifespan clinical studies. Due to these confounders, using the termi- of a mouse is about 2 years, compared to about 75 years in nology of genetics, any genes associated with atherosclero- humans. Mice weigh much less, about 30 grams for the sis will therefore display incomplete penetrance with the adult. The lipid profile in the mouse is very different from phenotype of atherosclerosis. Thus there is a great need to that in humans, who carry about 75% of their plasma cho- develop animal models to discover the root causes of, and lesterol on LDL. Mice carry most of their cholesterol on genes that are associated with, this disease. high-density lipoprotein (HDL), which we know in humans The modern theory of atherogenesis, championed by is protective against atherosclerosis. Thus, mice fed their Russell Ross, is the “response to injury hypothesis,” which normal low-fat chow diet do not get atherosclerosis, while states that atherosclerosis is an excessive fibroproliferative it is a common disease in humans. One difference, which is response to an insult upon the arterial wall (1). In a typical an advantage of all animal models, is the ability to control scenario of human atherogenesis, plasma-derived lipopro- the environment and diet in mouse studies, which is im- teins are retained in, and modified by, the arterial wall. possible for long-term human studies. Human genetic stud- These modified lipoproteins are inflammatory and, by a ies are limited in range to various types of association stud- mechanism not completely understood, lead to endothelial ies. With mice, on the other hand, many additional kinds of cell activation and expression of cell-surface adhesion mol- genetic experiments are possible, including breeding and ecules. Blood monocytes stick to these adhesion molecules genetic engineering. and then traverse the endothelium into the artery wall, There are many advantages of using mice for experimen- where they differentiate into macrophages. The macro- tal atherosclerosis research, including their relative ease pages then take up modified lipoproteins via scavenger re- and thriftiness to acquire and maintain. Their generation ceptors and thus become cholesterol-engorged foam cells. time is short, at about 9 weeks, 3 weeks for gestation and about 6 weeks until sexual maturity. It is easy to breed very large cohorts for experimental studies, and mice can The Rockefeller University, New York, New York 573 Vol 48, No 6 Laboratory Animal Science December 1998 develop atherosclerosis in a very short timeframe, as dis- diet contained 30% fat, 5% cholesterol, and 2% cholic acid, cussed below. Classical genetics in the mouse is very well and led to atherosclerosis in certain strains of inbred mice established and is aided immensely by the availability of such as C57BL6 (3). However, other strains were resistant hundreds of inbred strains. Many of these inbred strains to this diet. The F1 hybrids created by cross breeding ath- were created by lay people, mouse fanciers who bred mice erosclerosis-susceptible and -resistant strains had inter- for interesting coat colors. Thus, many genes have been iso- mediate levels of atherosclerosis, implying that genetic fac- lated in the homozygous state in these inbred strains. With tors were involved in determining atherosclerosis suscep- the coming of age of molecular genetics, it is now possible to tibility on this diet (4). However, this was still a very toxic add exogenous transgenes into mice, which can also be done diet on which the mice lost weight and often got sick with in many other species. However, uniquely in mice, it is also morbid respiratory infections. Beverly Paigen and her col- possible to knock out or replace endogenous genes; this is leagues modified the Thomas-Hartroft diet by blending it one of the main advantages of working in the mouse model. one part to three parts with a 10% fat diet to yield what is The major disadvantage of the mouse model is their small called by many scientists today the “Paigen diet,” which size, which makes it difficult but not impossible to perform consists of 15% fat, 1.25% cholesterol, and 0.5% cholic acid surgical manipulations and in vivo imaging. But there have (5). Paigen studied 10 different inbred mouse strains on been recent advances in these techniques that have over- this diet and found a large variation in atherosclerosis sus- come many of the size limitations, such as the ability to ceptibility; this variation did not correlate with the total perform imaging of abdominal atherosclerotic lesions in plasma cholesterol levels, implying that other factors were living mice, cardiac catheterization to determine cardio- involved in susceptibility to this diet. vascular function in free-ranging mice, and surgical liga- Paigen and her colleagues also developed assays that are ture of coronary arteries giving rise to myocardial ischemia. widely used to quantify atherosclerosis in the mouse model. What then are some of the uses for mouse models of ath- The most standard assay is the measurement of the cross- erosclerosis? First, this model is useful for the identifica- sectional lesion area in the aortic root (6). In this assay, tion of atherosclerosis susceptibility-modifying genes. Many freshly perfused and isolated hearts are fixed in formalin, humans who develop atherosclerosis have only moderate embedded in gelatin, frozen, and cut into thin sections at cholesterol and no other known risk factors. Thus, by using anatomically defined sites in the aortic sinus and valve various animal models it may be possible to identify genes region. These sections are stained for lipids, and the lesion that modify atherosclerosis susceptibility, and if this infor- area is measured microscopically. mation translates to humans, it will help in the identifica- Although this model has been widely employed and is tion of individuals with a genetic predilection toward ath- of significant use in the study of atherosclerosis, the pa- erosclerosis. These subjects can then be treated aggressively thology of the lesions are not ideally suited as a model for to reduce environmental and behavioral factors (e.g., diet, human atherosclerosis. This shortcoming led many inves-